Project Summary Hypertension is a major risk factor for heart failure, stroke, and kidney disease. Even small, but persistent increases in arterial blood pressure lead to pathological remodeling of the vessels and heart, and are associated with reduced life expectancy. Vascular smooth muscle cell (VSMC) contractility and remodeling are two critical Ca2+-dependent mechanisms that contribute to hypertension. The highly Ca2+-selective plasma membrane (PM) Orai channel proteins which are controlled by the universal endoplasmic reticulum (ER) Ca2+ sensor protein, STIM1 are upregulated in VSMC during hypertension. Our published and preliminary data demonstrate that Store-independent Orai channels (SICs) are encoded by heterohexamers of two exclusively mammalian proteins: Orai3 and the long variant of Orai1, Orai1?. We demonstrated that Orai1?/3-SIC is required for VSMC migration and for neointima formation after vascular injury, yet does not play a role in proliferation. Orai1?/3-SIC expression and activity are specifically upregulated in VSMC during hypertension. Our major objective is to determine the role and regulation of this unique Orai1?/3-SIC in VSMC dysfunction during hypertension. We hypothesize that Orai1?/3-SIC generate Ca2+ signals in VSMC that control unique downstream pathways to mediate contractility and/or remodeling in hypertension. We propose three aims. 1: To determine the specific heteromeric assembly and regulation of native Orai1?/3-SIC in VSMC. The oligomeric state and mode of regulation by Ca2+ of native Orai1?/3-SIC will be investigated using novel fluorescently-labeled Orai isoforms expressed at endogenous levels in Cas9/CRISPR Orai-knockout VSMC. These studies will combine high-resolution FRET and confocal imaging and biophysical channel measurements using mutant and concatenated Orai1?/3 hexameric channel constructs. 2: To elucidate the downstream SIC-specific signaling pathways and determine the spatial and temporal Orai1?/3-generated Ca2+ microdomains in migrating VSMC. Having established the role of Orai1?/3-SIC in migration, we will utilize novel light-activated Orai-derived single-molecule probes containing tethered genetically-encoded Ca2+ indicators, to define Orai1?/3-generated Ca2+ signals, their PM localization and the immediate signaling pathways and long-term transcriptional regulation in migrating VSMC. 3: To determine the in vivo role of Orai1? and Orai3 in vascular dysfunction, contractility and remodeling in hypertension. Using Orai1?SMC-/- and Orai3SMC-/- mice expressing ratiometric GCamp5/mCherry Ca2+ indicator exclusively in SMC, we will determine the specific role of VSMC Orai1?/3-SIC-mediated Ca2+ signals in hypertension and associated vascular dysfunction. This proposal will use mechanistic and translational approaches to enhance our understanding of VSMC physiology and pathophysiology, and establish VSMC Orai1? and Orai3 as novel therapeutic targets in hypertension and other vascular diseases.